PROJECT SUMMARY/ABSTRACT While C9ORF72 mutations were originally described in individuals with either amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTLD), the clinical spectrum is wider and includes patients clinically thought to have amnestic dementia compatible with Alzheimer's disease. Pathologically, while most c9FTD/ALS cases have Type B TDP-43 pathology, this is not universal and some cases also have Type A or even Type C TDP-43 pathology. The basis for this heterogeneity is unknown and will be explored in this project. Discovery of RAN translation and the generation of antibodies specific to predicted RAN translation products (C9RANT) from the GGGGCC hexanucleotide repeat in C9ORF72 is a major development in identifying disease specific biomarkers. Thus, C9RANT immunohistochemistry might prove to be a valuable tool for screening for C9ORF72 expansion carriers. The nature of the pathology related to RAN translation remains to be defined. Pathologic inclusions in c9FTD/ALS are heterogeneous and include TDP-43-positive and TDP-43-negative neuronal and glial inclusions. TDP-43 negative inclusions are detected with immunohistochemistry for ubiquitin (Ubq) and Ubq-binding proteins such as p62/sequestosome and ubiquilin2 (Ubqln2). It is uncertain how inclusions detected by C9RANT immunohistochemistry relate to those detected by Ubq, Ubqln2 and p62. The relative contribution of these various pathologic processes to the clinical phenotype is an area that remains unexplored and will be addressed in this project using qualitative and quantitative methods, including advanced digital microscopy, as well as immunogold electron microscopy. It also remains to be determined if genetic risk factors, in particular the C9ORF72 GGGGCC repeat length, influence clinical and pathologic heterogeneity of c9FTD/ALS. The following Specific Aims are proposed to address gaps in knowledge about c9FTD/ALS. Aim 1. Characterize the multiproteinopathy of c9FTD/ALS using double labeling immunohistochemistry and immunogold electron microscopy with antibodies specific to c9RAN, Ubqln2, TDP-43, p62 and to markers of proteotoxic stress. Aim 2. Map distribution of neuronal inclusions immunoreactive with antibodies to c9RAN polypeptides in all reading frames in sense and antisense direction, as well as arginine methylation, with markers of neurodegeneration using digital microscopy. Aim 3. Create a database of quantitative and semiquantitative data from Aims 1 and 2 as well as biochemical data from Project 2 for exploratory associations with clinical features as well as C9ORF72 repeat length (from Project 1) and common genetic variants in GRN and TMEM106B (from Core B).